Leishmania organisms are intracellular protozoan parasites of macrophages that cause a wide range of clinical diseases in humans and domestic animals, primarily dogs. In some infections, the parasite may lie dormant for many years. In other cases, the host may develop one of a variety of forms of leishmaniasis. For example, the disease may be asymptomatic or may be manifested as sub-clinical visceral leishmaniasis, which is characterized by mild symptoms of malaise, diarrhea and intermittent hepatomegaly. Patients with sub-clinical or asymptomatic disease usually have low antibody titers, making the disease difficult to detect with standard techniques. Alternatively, leishmaniasis may be manifested as a cutaneous disease, which is a severe medical problem but is generally self-limiting, or as a highly destructive mucosal disease, which is not self-limiting. Finally, and most seriously, the disease may be manifested as an acute visceral infection involving the spleen, liver and lymph nodes, which, untreated, is generally a fatal disease. Symptoms of acute visceral leishmaniasis include hepatosplenomegaly, fever, leukopenia, anemia and hypergammaglobulinemia.
Leishmaniasis is a serious problem in much of the world, including Brazil, China, East Africa, India and areas of the Middle East. The disease is also endemic in the Mediterranean region, including southern France, Italy, Greece, Spain, Portugal and North Africa. The number of cases of leishmaniasis has increased dramatically in the last 20 years, and millions of cases of this disease now exist worldwide. About 2 million new cases are diagnosed each year, 25% of which are visceral leishmaniasis. There are, however, no vaccines or effective treatments currently available.
Diagnosis of Visceral leishmaniasis can not always be made only on the basis of clinical symptoms because visceral leishmaniasis shares its clinical features with other diseases such as malaria, typhoid fever and tuberculosis occurring commonly in the same endemic areas. Thus, the diagnosis of visceral leishmaniasis largely relies on parasitological or serological methods. The former is microscopic detection of amastigotes in aspirates of spleen and bone marrow or detection of promastigotes through cultivation of the aspirates. Unfortunately, this method requires the biopsy of bone marrow, liver, spleen, or lymph nodes may be required, which may lead to secondary infection or further disfigurement of the patient. Additionally, along with being an invasive diagnosis, it takes a long period of time to diagnose and results are commonly non-conclusive. Therefore, this method is invasive, time-consuming, and not sufficiently sensitive, thereby rendering it inefficient.
Less invasive and time consuming laboratory procedures do exist, however, these procedures suffer from either lack of sensitivity or cumbersome implementation. For example the Liquid Direct Agglutination Test (LQ DAT: Ahfad University, Khartoum and IPB, Addis Abbeba) and the Freeze Dried DAT (FD DAT: Meredith et al. 1995) have good sensitivity, but require multiple pipetting steps and incubation, which makes implementation of this test difficult in developing countries. Similarly, the Latex Antigen Agglutination Test in Urine (KATEX®: Kalon Biological Ltd-UK) has good sensitivity and specificity, but requires a cumbersome urine boiling step and further suffers from low reproducibility. Finally, the rK39 and rK26 dipstick tests (Inbios®, Seattle, Wash.) can rapidly give results within a matter of minutes, but these tests lack sensitivity in certain geographic regions such as Sudan, Ethiopia and Kenya. Because the dipstick tests can be easily, quickly, and affordably implemented, yet suffer from lack of antigen sensitivity, discovery of new antigens is necessary for more accurate diagnosis of leishmaniasis.
Among defined leishmanial antigens reported previously, rK39 appears to be the best antigen for serodiagnosis of visceral leishmaniasis in terms of both sensitivity and specificity. rK39 is sensitive and reliable even on a strip format, which is feasible for field use, and the rK39 strip test has high sensitivity in India, Nepal and Brazil. In Sudan, Ethiopia and Kenya, however, the sensitivity of the strip test falls to 67% and the negative responses on the strip test appear to correlate with lower reactivity by ELISA. Thus, new diagnostic antigens are needed to complement rK39 to contribute to the development of a more accurate diagnosis of leishmaniasis. The present invention fulfills these needs and many other related needs.